Gold grid

To prepare cryo EM grids, 4 mg/ml full-length G614 variant spike trimer^{37 (link)} in DDM and 5 mg/ml 17B10–1 Fab (1:3 S protein trimer: Fab ratio) were first incubated at 4°C for 30mins. Four μl of the mixture was then applied to a 1.2/1.3 Quantifoil gold grid (Quantifoil Micro Tools GmbH), which had been glow discharged with a PELCO easiGlow^™ Glow Discharge Cleaning system (Ted Pella, Inc.) for 60 s at 15 mA. Grids were immediately plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific), and excess protein was blotted away by using grade 595 filter paper (Ted Pella, Inc.) with a blotting time of 4 s, a blotting force of −12 at 4°C with 100% humidity. The grids were first screened for ice thickness and particle distribution. Selected grids were used to acquire images by a Titan Krios transmission electron microscope (ThermoFisher Scientific) operated at 300 keV and equipped with a BioQuantum GIF/K3 direct electron detector. Automated data collection was carried out using SerialEM^{38 (link)} version 3.8.6 at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.825 Å) at an exposure rate of 27.448 electrons per pixel per second. Each movie added a total accumulated electron exposure of ~54.442 e-/Ų, fractionated in 52 frames. Data sets were acquired using a defocus range of 0.8–2.2 μm.